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Cell metabolism reprogramming method and application thereof

A technology of cell metabolism and reprogramming, applied in the direction of microorganism-based methods, applications, botany equipment and methods, etc., can solve the problem of high cost and achieve low-cost effects

Pending Publication Date: 2022-05-03
SHANGHAI JIAO TONG UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, there are few methods for studying the interaction of multiple genes at a global level, and the cost is high

Method used

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  • Cell metabolism reprogramming method and application thereof
  • Cell metabolism reprogramming method and application thereof
  • Cell metabolism reprogramming method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Example 1. Construction of a tool plasmid for a double sgRNA library

[0054] Such as figure 2 As shown, two sgRNA expression cassettes were constructed on the pE1A plasmid, one expression cassette contains the J23100 constitutive promoter, N20, gRNA scaffold and T0 terminator.

[0055] Then insert a toxic protein ccdB in the middle of the two expression frames, and introduce the restriction site BbSI of Golden Gate at the same time, and add the recognition site and protective base of BbSI to the primers SEQ ID NO.3 and SEQ ID NO.4 in the tool plasmid.

[0056] The specific sequence is as follows:

[0057] NO.3: 5-GATCGAAGACGTGTTTTAGAGCTAGAAATAGCAAGT-3;

[0058] NO.4: 5-GATCGAAGACACTAGTATTATACCTAGGACTGAG-3.

[0059] Using the principle of homologous recombination, through the above primers, two sgRNA expression cassettes, toxic protein ccdB and BbSI restriction sites and recognition sites were simultaneously introduced into the simultaneous pE1A plasmid by the meth...

Embodiment 2

[0060] Embodiment 2, construct the recombination fragment of double sgRNA library

[0061] as attached figure 2 In the technical route shown, first, n pairs of primers for constructing the corresponding library are synthesized, and then the fragments of the double sgRNA library are amplified by PCR using the plasmid containing the two sgRNA expression cassettes in Example 1 as a template.

[0062] In the n pairs of primers, except for the N20 part of the sequence (marked as NNN in the sequence), the other sequences are the same, and the N20 part of the sequence is designed according to different library construction genes. Specific primer sequences such as SEQ ID NO.3 and SEQ ID NO.4:

[0063] NO. 3: 5-GATCGAAGACACACTAGTNNNGTTTTAGAGCTAGAAATAGCAAGT-3; NO. 4: 5-GATCGAAGACTAAAACNNNAACTAGTATTATACCTAGGACTGAG-3.

[0064] After the fragments of the library were amplified by PCR, electrophoresis was performed on the agarose gel. After running the gel, the gel with the correct band ...

Embodiment 3

[0065] Embodiment 3, construct the plasmid of double sgRNA library

[0066] After the recombinant system of the double sgRNA library was obtained in Example 2, the recombinant system was constructed into Escherichia coli E.coli K12 by chemical transformation, and then the recombinant system was coated to have screening resistance (in this example, 5 μg / mL tetracycline ) on a solid plate, so that it grows about 10n 2 transformants (it has been verified that each plate can have about 1000 transformants).

[0067] Then it was eluted with LB liquid medium, mixed evenly, added 15% glycerol and placed in a -80°C refrigerator for storage. The obtained double sgRNA library plasmid strain is obtained, and then the obtained strain is used as the seed bacteria, and after expanding in LB medium containing 5 μg / mL tetracycline, the plasmid is extracted, which is the plasmid of the constructed double sgRNA library.

[0068] Taking this example as an example, a dual sgRNA library targeting...

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Abstract

The invention discloses a cell metabolism reprogramming method and application thereof, and belongs to the field of genetic engineering.The cell metabolism reprogramming method utilizes sensitive nodes of a cell metabolism network to reprogram the metabolism network and mainly comprises the three parts of CRISPRi-based double-gene combination weakening, cell high-throughput screening and survival rate-based gene interaction analysis. The invention provides an intelligent metabolism reprogramming method which is novel, simple, efficient and low in cost, and can be widely used for operating a cell metabolism network.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a method for cell intelligent metabolic reprogramming. Background technique [0002] As an interdisciplinary field of engineering and science in the 21st century, engineering biology is of great help to the exploration of the basic laws of life activities and breakthroughs in biotechnology. It enables us to design and construct novel biomolecular assemblies, networks and pathways to reprogram organisms. Its core goal is to apply engineering biotechnology to precisely control, regulate and reshape the metabolic network of microbial cell factories to achieve the synthesis of desired products. [0003] The control, regulation and remodeling of cellular metabolic networks is also called metabolic reprogramming, which was first widely discovered in the field of cancer. In the field of engineering biology or synthetic biology, metabolic reprogramming is the fundamental way to achie...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/74C12N15/65C40B50/06C40B40/02C12N1/21C12N15/54G16B35/20G16B35/10G16B25/20G16B30/10C12R1/63
CPCC12N15/74C12N15/65C40B50/06C40B40/02C12N9/12C12Y207/13003G16B35/20G16B35/10G16B25/20G16B30/10
Inventor 陶飞谭春林许平
Owner SHANGHAI JIAO TONG UNIV
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